Lung Metastasis Of Breast Cancer Related Genes

Part I: Proteomic Analysis of Differential Proteins Expression in Human Breast Cancer Cell Lines with Differently Lung Metastatic PotentialPurpose: We have developed a well established highly lung metastatic potential breast cancer cell line, MDA-MB-435HM. In present study, we try to apply proteomic analysis on MDA-MB-435HM and its parental cell to find out the differentially expressed protein and, furthermore, to study the clinical significance of some proteins of them.Methods: 2-DE coupled with LC-IT-MS were applied to MDA-MB-435HM and its parental cell MDA-MB-435. We use SEQUEST to reveal the differentially expressed proteins. The mRNA and protein levels of 3 of the differentially expressed proteins were confirmed by real-time RT-PCR and Western blot analysis. Eighty breast cancer samples were accessed by immunohistochemistry stain with antibody of these 3 proteins to investigate their clinical significance.Results: Totally, we found 11 proteins expressed differently between two cells by proteomics analysis, which includes PDX6, CRAB, HSP27, TPM1-4, 14-3-3 protein epsilon, HSP60, tumor protein D54 and Cathepsin D precursor. Real-time quantitative RT-PCR analysis demonstrated that the mRNA level of CRAB, HSP60 and PDX6 in highly metastatic potential breast cell line were 0.24, 3.15 and 2.43 folds of its parental generation, respectively. Western blot revealed that CRAB was only expressed in MDA-MB-435 cell lines. While, the protein level of HSP60 and PDX6 were upregulated in highly metastatic potential breast cell line. The positive rates of CRAB between patients without axillary lymph node metastasis and patients with axillary lymph node metastasis were 52.5% and 32.5%, respectively (P=0.113). The positive rates of HSP60 between 2 groups were 32.5% and 72.5%, respectively(.P=0.001). The positive rates of PDX6 between 2 groups were 35.0% and 2.5%, respectively (P=0.002).Conclusions: The expression of HSP60 and PDX6 were up-regulated in highly lung metastasis breast cancer cell MHA-MB-435, and may associated with invasion in breast cancer. Further studies were needed to investigation the underling mechanisms. These data provide the basis for searching for potential markers of breast cancer prognosis and give some clues to elucidate the mechanism of breast cancer metastasis.Part II: Trophinin and the potential of invasion and metastasis of breast cancerObjective: To explore the expression of trophinin between breast cancer cell lines with different lung metastatic potential and its possible roles in breast cancer proliferation and metastasis.Methods: We have conduceted immunofluorescence stain to determine the subcellular location of trophinin in breast cancer cell line MDA-MB-435. RT-PCR was applied to analysis the mRNA level of trophinin of MCF7 and MDA-MB-435 after treated with estradiol (E2). Real-time RT-PCR were conducted to assess the mRNA level of trophinin between highly lung metastatic potential breast cancer line MDA-MB-435HM and its parental cell line. Expression vector pcDNA3.1(+)-trophinin was developed based on the vector pcDNA I-trophinin to upregulate the level of trophinin in breast cancer cell line MDA-MB-435. WST was performed to evaluate the proliferation rate of MDA-MB-435, MDA-MB-435-mock and 2 clones of trophinin transfectants. The flow cytometry analysis were used to evaluate the S phage of transfectants and its parental cells. Matrigel was used to evaluate the invasion potential of breast cancer cell line MDA-MB-435 after transfection of trophinin in vitro. RT-PCR was porfermed to determined the changes of mRNA level of cyclins, VEGF, bFGF and MMPs et al. after the transfections. Trophinin transfectants, mock transfectants and their parental cells were implanted orthotopically as intact tissue into nude mice to further test the results from in vivo study.Results: Trophinin was located at cytomembrane and cytoplasm in MDA-MB-435 cell. The mRNA level of trophinin in ER positive breast cancer cell line MCF7 was up-regulated by E₂, with maxim level at the concentration of 20nM. However, E₂ has no effect on ER negative breast cancer cell line MDA-MB-435. The mRNA level of trophinin in highly lung metastatic potential breast cacner cell MDA-MB-435HM is 3.61 folds of its parental cells. After transfection of trophinin, the mRNA level of 2 transfectant clones were upregulated compared with their parental cells. The cell proliferation rate did not increased after transfection of pcDNA3.1(+)-trophinin. Flow cytometry analysis revealded that the S phage rates were similar among MDA-MB-435, mock transfectant and 2 trophinin transfectant clones. The mean number of cells invaded through transwell was significantly higher in transfectant group (81/HF vs 157/HF). RT-PCR analysis revealed that the mRNA level of MMP7 in transfectantgroup was higher than non-trophinin transfectants. In vivo study indicated that the mean number of lung surface metastasis was significantly higher in transfectant group than in non-trophinin transfectants group (MDA-MB-435 vs mock vs clone 1 vs clone 2: 26.3/lung vs 24.5/lung vs 53.8/lung vs 54.4/lung).Conclusion: Trophinin expression was associated with invasiveness of breast cancer. Overexpression of trophinin leads to a more invasive phenotype of breast cancer cells, which might be through its possible roles in upregulating level of MMP7. Further investigation in this aspect is proposed to make clear the exact meachanisms of trophinin gene on invasion and metastasis of breast cancer.